The ability of the adaptive immune system to respond to pathogenic infections depends on the diverse arrays of antigen receptors expressed by T and B lymphocytes. In human and other vertebrates, V(D)J recombination is the primary mechanism by which the antigen receptor diversity is generated. The recombination reaction goes through distinct steps, involving the generation and repair of DNA double strand breaks (DSB). V(D)J recombination is tightly regulated both before and after DNA cleavage. Before cleavage, the two rearranging gene segments must be accessible and brought together for concerted cleavage. After cleavage, DSB must be repaired efficiently and properly to avoid cell cycle arrest and disruption of genome integrity. Evidence suggests that transcriptional enhancers are involved in both types of regulation by targeting specific gene segments for cleavage and by promoting efficient repair of DSB. This application focuses on the role of the transcriptional enhancer at the T cell receptor beta locus in post-cleavage steps of the recombination reaction. Specifically, we propose to determine (i) the precise steps at which enhancer mutation impairs the post-cleavage steps of the recombination reaction; (ii) the Imechanisms by which the enhancer normally promotes the repair of coding end DSB; and (iii) the mechanisms by which the enhancer mutation accelerates the development of thymic lymphomas in p53-deficient mice. Defects in V(D)J recombination could result in immunodeficiencies as well as lymphoid malignancies. The proposed studies may help elucidate the role of enhancers in regulating DSB repair, V(D)J recombination, genome instability, and tumorigenesis.